Antibodies, also known as immunoglobulins (Ig), are glycoproteins that play a central role in immune responses. IgG antibodies (Abs) and fragments of IgG Abs have become major biotherapeutics for treating human diseases.
There are five functional classes of immunoglobulins, i.e., immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin E (IgE). Among them, IgG is the most abundant immunoglobulin in serum.
The variable region (Fab) of the antibody molecule is involved in direct binding of the target antigen. All naturally-occurring antibodies each include a constant domain known as the Fc (Fragment, crystallizable) region, which is composed of constant domains depending on the class of the antibody. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils. (Woof J, Burton D (2004) Nat Rev Immunol 4 (2): 89-99; Heyman B (1996) Immunol. Lett. 54 (2-3): 195-199). Glycosylation of the antibody Fc fragment is essential for Fc receptor-mediated activity. (Peipp M. et al., Blood (2008) 112(6):2390-2399).
The constant (Fc) domain of naturally-occurring antibodies is usually N-glycosylated. The linkage of carbohydrates to proteins occurs through N-linked glycosylation—the attachment of a sugar to the amide nitrogen atom on the side chain of asparagines. The linkage to amino acids is generally embedded in a conserved sequence of amino acids. Glycosylation in the Fc region significantly affects Fc effector functions such as activation of complement (Roos et al., J Immunol. (12):7052-7059.2001) or binding to receptors via their constant (invariable sequence) domains (Mimura et al., J Biol. Chem. 2001 Dec. 7; 276(49):45539-45547; Shields et al., J Biol. Chem. 276(9):6591-6604 (2001)).
It has been found that the sequences of the oligosaccharides attached to Fc regions are essential to the stability and function of the antibodies. For example, the addition of sialic acid (SA), sialylation, at Asn 297 on the Fc domain and terminal galactosylation modify the anti-inflammatory properties of immunoglobulins and play a role in rheumatoid arthritis (RA). (Kaneko, Y et al., Science. (2006) 313(5787):670-673).
Human antibodies prepared by conventional methods, such as genetic engineering, comprise mixtures of different glycoforms, i.e., containing different oligosaccharides attached to their Fc regions.
The clinical successes of the antibodies rituximab and alemtuzumab for hematological malignancies, and trastuzumab for solid breast tumors, have demonstrated that human or humanized monoclonal antibodies can be useful drugs for the treatment of cancer. All of these antibodies have been approved and are currently in the market. Yet, a need remains for improving this kind of drugs. For instance, rituximab induces a response only in approximately 60% of the patients with relapsed/refractory low-grade non-Hodgkin's lymphoma.
The preferred type of monoclonal antibody for target-cell killing applications is an IgG1-type mAb, where at least the constant (or the Fc) region is of human origin (i.e., chimeric, humanized, or fully-human IgG1s). IgG1 mAbs can exert their therapeutic effects via three classes of mechanisms. Direct binding of the antibody via its variable region to the target molecule on the surface of cancer cells can lead to cell death or inhibition of tumor growth, for example by triggering apoptosis upon cross-linking of target molecules by the antibody. The Fc region operates in the other two-types of mechanisms: complement-mediated cytotoxicity, and Fcγ receptor (FcγR)-dependent effector functions. Some Fc effector functions of IgG1s are mediated by the interactions of the Fc region of target-cell bound antibody molecules and Fcγ receptors on the surface of immune cells. The Fc domains of immunoglobulins have been shown to have effector functions which are primarily complement fixation and Fc receptor (FcR) binding. appropriate glycosylation at the conserved glycosylation site (N297) of the Fc domain is essential for the efficient interactions between mAbs and Fc receptors (FcR) and for the FcR-mediated effector functions, including antibody-dependent cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Fc receptors bind to the constant domains of immunoglobulins and a number of receptors have been defined that are thought to mediate accessory functions including opsonization and ADCC (Daeron, M. Annual Review Immunology, (1997) 15; 203-234, Ravetch and Clynes, Annual Review of Immunology. 1998. 16:421-432).
The conserved glycosylation site on the Fc region of antibodies is a target for modulation of antibody effector functions. The crystal structure of a biosynthetic intermediate of human IgG1, bearing immature oligomannose-type glycans and reported to display increased antibody-dependent cellular cytotoxicity, has shown that glycan engineering can bias the Fc to an open conformation primed for receptor binding. (Crispin M., et al., J. Mol. Biol. 387(5):1061-1066 (2009).)
More specifically, certain glycoforms of a human antibody exhibit improved therapeutic effects while others possess undesired properties. For example, de-fucosylated but glycosylated Herceptin® is at least 50-fold more active in the efficacy of Fcγ receptor IIIa mediated ADCC than those with alpha-1,6-linked fucose residues (Shields, R L, et al. J. Biol. Chem., Vol. 277(30):26733-26740, (2002)). Similar results were reported for Rituximab® and other mAbs (Shinkawa T, et al., J. Biol. Chem. (2003) 278:3466-3473).
Strome et al., (WO 2007/146847) disclose a method of preparing antibodies with homogeneous glycosylation states by first deglycosylating an antibody or antibody fragment and then attaching a chemically synthesized sugar to the protein. This method lacks the ability to control the glycosylation process and does not enable control over the conjugation of specific carbohydrates at specific sites during the formation of a desired glycoform. Thus, there is a need for methods of preparing human antibodies comprising desired glycoform(s) wherein the glycosylation is performed in a stepwise manner so as to improve their therapeutic efficacy.